Bioorganic & Medicinal Chemistry 18 (2010) 6987–6994

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Bioorganic & Medicinal Chemistry

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Identification of small molecule inhibitors of telomerase activity throughtranscriptional regulation of hTERT and calcium induction pathwayin human lung adenocarcinoma A549 cells

Yerra Koteswara Rao a, Te-Yu Kao b, Ming-Fang Wu c,d, Jiunn-Liang Ko c,e,*, Yew-Min Tzeng a,*

a Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Wufeng, Taiwan, ROCb Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, ROCc Department of Medical Oncology and Chest Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROCd School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROCe Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 July 2010Revised 9 August 2010Accepted 10 August 2010Available online 13 August 2010

Keywords:Small moleculeTelomerase activityhTERThTERT promoterAnchorage-independent growthCalcium release

0968-0896/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.bmc.2010.08.021

* Corresponding authors. Tel.: +886 4 24730022x1(J.-L.K.); tel.: +886 4 23323000x4471; fax: +886 4 233

E-mail addresses: jlko@csmu.edu.tw (J.-L. Ko), yTzeng).

High telomerase activity (TA) is detected in most cancer cells; and thus, TA inhibition by drug or dietaryfood components is a new strategy for cancer prevention. In this report, we examined the effects offourteen natural or synthetic compounds on TA in human lung adenocarcinoma A549 cells. The resultsdemonstrated that some of the tested compounds inhibited TA, being 20-hydroxy-2,3,40,60-tetramethoxy-chalcone (HTMC) was the most potent among tested. In A549 cells, HTMC also inhibited the cell prolifer-ation, decreased the expression of human telomerase reverse transcriptase (hTERT) and sequentiallyreduced the hTERT promoter. In soft agar assay HTMC treatment reduced the colony formation of A549cells. Cellular fractionation and immunofluorescence assay demonstrated that there was no translocationof hTERT from nuclei to cytoplasm. Further studies revealed that the release of Ca2+ was the underlyingmechanism of suppressed TA and hTERT transcription in A549 cells exposed to HTMC. These in vitro datasupport the development of HTMC as a therapeutic agent for cancer complications.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Telomerase as an enzyme is responsible for the renewal of thechromosomal ends, the so-called telomeres.1 Telomeres are essen-tial units that prevent the loss of genetic information. In normal so-matic cells, which show little or no telomerase activity (TA) tosynthesize new telomeres.1 In contrast, most cancer cells havemechanisms that compensate for telomere shortening throughthe activation of telomerase, allowing them to stably maintain theirtelomeres and grow indefinitely.2 These observations suggest thattelomerase reactivation is a rate-limiting step in cellular immortal-ity and carcinogenesis, and telomerase repression can act as a tu-mor-suppressive mechanism.2 The key advantages of targetingtelomerase in comparison with most other cancer targets are its rel-ative universality, criticality and specificity for cancer cells, includ-ing the putative cancer stem cell.2 Telomere length in human isprimarily controlled by three major components including humantelomerase RNA component (hTR), telomerase-associated protein1 (TP1), and human telomerase reverse transcriptase (hTERT).2 Of

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1694; fax: +886 4 2475110195870 (Y.-M.T.).mtzeng@cyut.edu.tw (Y.-M.

these, we are primarily interested in identification of compoundthat induced down regulation of TA via suppression of hTERTexpression, because the hTERT is a core telomerase protein andits expression limited to germinal and cancer cells.2 It is well knownthat the expression of dominant-negative hTERT or its siRNA re-duces the lifespan of human cancer cells by completely inactivatingtelomerase.2 Therefore, it is thought that suppression of hTERTexpression is an ideal strategy for the development of anticancerchemotherapeutics.1,3

The inhibition of TA has been approached through different strat-egies, ranging from anti-sense-based inhibitors to random screeningof synthetic and natural products.1 Among the latter, several com-pounds have been reported include rubromycins,4 alterperylenol,5

epigallocatechin gallate,6 anthraquinone derivatives,7–9 G-quadru-plex ligands.10–12 In our previous studies, we found that loss oftelomerase activity can be potentially favorable prognostic markerin lung carcinomas.13–16 We are currently undertaking the therapeu-tic assessment of medicinal mushroom Antrodia camphorata.17–20 Inparallel, we sought to identify potent TA inhibitor compounds toexpand the scope of bioactive agents and to gain insight into theiranticancer mechanism of action. To this end, here, we report thescreening of 14 natural or synthetic compounds on TA in human lungadenocarcinoma cell line A549 cells.

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2. Results

2.1. Inhibition of telomerase activity (TA) in A549 cells

In an effort to identify potential candidate to inhibit TA, we haveselected 14 natural or synthetic compounds (Fig. 1), based on theirgrowth inhibitory potential against tumor cell proliferation andinflammatory mediators production in different type of cell linesas we reported previously.21–26 The effects of these compounds(Fig. 1) on TA were examined in A549 cells with a random concentra-tion of 25 lM, using modified telomeric repeat amplification proto-col (TRAP) assay. We found that among tested, 20-hydroxychalconederivatives, 20-hydroxy-2,3,40,60-tetramethoxychalcone (HTMC)followed by compound 6 potentially inhibited TA with respect tocontrol (Fig. 2). Among the tested 30,40,50-trimethoxychalconederivatives, compounds 07–01, 07–02, 07–03, and isoflavonoid,isobonducellin (37) have moderate effect on TA (Fig. 2). In contrast,the flavonol glycosides, kaempferitrin (20) and kaempferol 3-O-b-D-glucopyranosyl-(1?4)-a-L-rhamnopyranosyl-7-O-a-L-rhamno-pyranoside (21); flavanones, 5,7-dimethoxyflavonone (31) and7-O-methylglabranin (32); lignan, phyllanthin (38); phenolic acid,4-methoxygallic acid (40); and 30,40,50-trimethoxychalcone deriva-tive 07–04 have no effect on TA at the tested concentration 25 lM(Fig. 2). Furthermore, the potent compound HTMC also inhibitedthe A549 cells TA in a dose (6.25, 12.5, 25.0 and 50.0 lM) and time(6 h, 12 h, and 24 h) dependent manner (see Supplementary data).Among the tested, HTMC was almost completely inhibited the TAof A549 cells at concentration of 25 lM (Fig. 2). Subsequently, HTMCwas used to examine the TA associated cellular and molecularevents in A549 cells.

We next examined the HTMC effect on A549 cell proliferationby MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The results demon-strated that HTMC treated for 24 h, A549 cells proliferation wasdecreased in a concentration-dependent manner (data not shown).Particularly, treated with 50 lM HTMC for 24 h reduced the A549cells proliferation by 59%.

2.2. Effect of HTMC on the expression of hTERT in A549 cells

Although active telomerase requires the co-expression of hTRand hTERT components, the expression of hTERT correlates closelywith TA in most cancers.2 Therefore, we attempted to elucidatewhether the above observed HTMC inhibitory effect on TA wasdue to the repression of hTERT expression in A549 cells. We usedRT-PCR, real-time RT-PCR (Taqman assay) and Western blot analy-sis and examined the changes in hTERT expression following A549cells treated with HTMC for 6 h. As indicated in Figure 3A, in HTMCtreated A549 cells the mRNA expression levels of hTERT decreasedin a dose-dependent manner. In contrast, hTR and GADD153 mRNAexpressions were not altered, demonstrated the specificity forhTERT mRNA (Fig. 3A). To quantify the degree of the decreasedhTERT mRNA, real-time PCR was performed using total RNA fromA549 cells treated with vehicle or HTMC in the concentration rangefrom 6.25 lM to 50 lM. As shown in Figure 3B, hTERT mRNA wasdecreased in a dose-dependent manner and reached to 92% after50 lM HTMC treatment for 6 h. Furthermore, the expression levelsof hTERT were also shown to undergo a dose-dependent decreasein whole cell extraction fraction following treatment with HTMCas confirmed by Western blot analysis (Fig. 3C).

2.3. Effect of HTMC on the hTERT promoter activity

The hTERT promoter activity has been closely associated withTA and hTERT expression in cancer cells but not in telomerase-neg-

ative normal cells.2 To determine whether the decreased levels ofhTERT in A549 cells was the result of decreased hTERT promoteractivity, we dissected the full-length hTERT fragment (�548 to+50) into five regions p548 (�548 to +50), p212 (�212 to +50),p196 (�196 to +50), p177 (�177 to +50) and p95 (�95 to +50)(Fig. 4A). After transfection, A549 cells were treated with 25 lMHTMC for 24 h and luciferase reporter activity was determinedby transient transfection assay. A plasmid expressing the bacterialb-galactosidase gene was co-transfected in each experiment toserve as internal control of transfection efficiency. The luciferaseactivity in HTMC treated cells was clearly down-regulated in theconstructs p548, p212, p196, p177, and p95 in the range approxi-mately from 25% to 70% as compared with untreated cells (Fig. 4B).

2.4. Effect of HTMC on the anchorage-independence of A549cells

We next examined the effect of HTMC on the colony formationefficiency of A549 cells using anchorage-independence cell growthassay. A549 cells are highly tumorigenic and readily form coloniesin soft agar medium. In this report, the results of soft agar assaydemonstrated that colonies formation efficiency of HTMC treatedA549 cells was drastically reduced as compared with untreatedcells. Additionally, the colonies observed in the HTMC-treated cul-tures were smaller and more densely packed than the untreatedcontrols (Fig. 5A). The number of colonies formed in agar was re-duced by 23% when A549 cells were stimulated with 6.25 lMHTMC. Stimulation of A549 cells with 12.5 and 25.0 lM HTMC de-creased the number of colonies by 96% and 99%, respectively(Fig. 5B) as compared with control cells. In general, increased theconcentration of HTMC pretreatment concomitantly reduced thecolony formation efficiency of A549 cells.

2.5. HTMC suppressed TA and hTERT expression in A549 cellsthrough calcium induction

Previous studies have shown that accumulation of wild-typeproteins in endoplasmic reticulum leads to release of Ca2+ fromorganelles that can be blocked by Ca2+ chelators.2,14 To evaluatethe signaling pathways involved in HTMC telomerase regulation,the intracellular calcium levels were analyzed in HTMC treatedA549 cells. As shown in Figure 6A, HTMC treated A549 cells re-leased the Ca2+ in a concentration-dependent manner, which wasdetected by calcium-sensitive indicator Fluo-3 AM. This was evi-denced by increased green staining in A549 cells after treatmentwith HTMC for 24 h (Fig. 6A). These results suggested that HTMCinduced the release of Ca2+ in A549 cells. To examine the specificrole of HTMC in the released Ca2+, A549 cells were pretreated withcalcium chelator BAPTA-AM for 1 h and then with HTMC for 24 h.Analysis of calcium fluorescence demonstrated that the influx ofcalcium release was decreased, indicated that HTMC significantlycontributed in the released Ca2+ in A549 cells (Fig. 6A). In addition,the specific role of Ca2+ released by HTMC on the regulation of TAwas also examined. For this, A549 cells were treated with 25 lMHTMC, or pretreated for 1 h with various cancer cells growth inhib-itors such as MEK inhibitor U0126 (10 lM), or ERK inhibitorPD98059 (20 lM), or MAPK inhibitor SB203580 (50 lM), or intra-cellular calcium chelator BAPTA-AM (20 lM), or 26S proteasomeinhibitor MG132 (20 lM), or nuclear export inhibitor leptomycinB (20 nM), or PI3 K inhibitor LY294002 (50 lM) and then treatedwith 25 lM HTMC for 24 h and assayed for TA using TRAP method.Similar to the previous results, HTMC inhibited the TA; however,the concomitant addition of 20 lM BAPTA-AM completely abol-ished the ability of HTMC to inhibit TA (Fig. 6B). Using TRAP andRT-PCR assays, we found that BAPTA-AM completely abolishedthe ability of HTMC to repress TA and hTERT mRNA levels in

Figure 1. Chemical structures of natural or synthetic compounds tested on telomerase activity in human lung cancer cell line A549 cells.

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A549 cells (Fig. 6C and D). In the absence of HTMC treatment, incu-bation of A549 cells with BAPTA-AM alone did not significantlychange the levels of TA and hTERT as compared with controls(Fig. 6C and D). Furthermore, immunofluorescence staining of25 lM HTMC treated A549 cells demonstrated a decreased expres-sion of hTERT, and there was no translocation of hTERT from nuclei

to cytoplasm (Fig. 6E). However, A549 cells pretreated for 1 h withintracellular calcium chelator BAPTA-AM and then treated with25 lM HTMC for 24 h, partly restored the expression of hTERT inthe nuclei (Fig. 6E). Taken together, these results suggested thatHTMC suppressed telomerase activity and hTERT expression byinducing calcium release in A549 cells.

Figure 2. Effect of various natural and synthetic compounds on the level of telomerase activity in human lung A549 cells. After (24 h) plating, cells were exposed to 25 lM ofeach tested compound for 24 h. Cell pellets were collected and subjected to TRAP assay. The telomerase activity in untreated A549 cells was as control (C). N, negative controlused lysis buffer only.

Figure 3. Effect of 20-hydroxy-2,3,40 ,60-tetramethoxychalcone (HTMC) on the expression of hTERT, hTR and GADD153 mRNA and protein level in A549 cells. (A) After 6 h ofincubation with HTMC, total RNA was isolated, and RT-PCR was performed using indicated primers, the amplified products were run in 1.5% agarose gel and visualized byethidium bromide staining. b-Actin was used as an house-keeping control gene. (B) A549 cells were treated with indicated concentrations of HTMC for 6 h, relative hTERTmRNA level was determined by real-time RT-PCR (Taqman Assay) and normalized to GAPDH mRNA. Representative data were from three independent experiments and *

indicates p <0.05 as compared with untreated cells. (C) Western blot analysis of hTERT subunit A549 cells treated with indicated concentrations of HTMC for 24 h.

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3. Discussion

The unique role of telomerase in particular in cancer cells intro-duced a new area of research directed to the goal of telomeraseinhibition as smart approach for cancer therapy.1 Numerous stud-ies using various active ingredients were performed to inhibit TA.Beside the G-quadruplex ligands,10–12 small synthetic and nucleicacids based telomerase inhibitors, there is a multiplicity of com-pounds that can cause a decrease in TA.1 Differentiating agents likeall-trans retinoic acids, adriamycin and doxorubicin reduced TAand hTERT mRNA expression.1 Another family of telomerase inhib-itors is the isothiazolone derivatives which could reduce the en-zyme activity by interfering with a cysteine in its active center.1

Other putative inhibitors derived from natural sources are curcu-min, epigallocatechin gallate (EGCG), retinoic acids and a herbalcomplex based on plant extracts from Hoelen, Angelicae, Scutellar-riae, Glycyrrhizae radix and other herbs.1 Previous studies indicatethat EGCG reduce telomerase activity by decrease in hTERT pro-moter methylation and increase in hTERT repressor E2F-1 bindingat the promoter.27 Our previous report also demonstrated that cur-cumin inhibit telomerase activity and its transcription factorsincluding NFkB, AP1 and Sp1.16 Multiple mechanisms and severaltranscription factors regulate telomerase ability at DNA and pro-tein level. In this report, our efforts attempted to identify potentialcompound that can modulates TA by up-regulating calcium con-centration and repress hTERT expression at transcriptional level.

Figure 4. Effect of 20-hydroxy-2,3,40 ,60-tetramethoxychalcone (HTMC) on hTERT promoter activity. A549 cells were transfected with luciferase reporter plasmid containinghTERT promoter deletion mutants p548 (�548 to +50), p212 (�212 to +50), p196 (�196 to +50), and p177 (�177 to +50), and were left untreated or treated with HTMC for24 h followed by measurement of hTERT activity by luciferase reporter assay.

Figure 5. Effect of HTMC on anchorage-independent growth. (A) A549 cells weretreated with different concentrations of 20-hydroxy-2,3,40 ,60-tetramethoxychalconein 0.35% agarose containing 10% FBS-DMEM over 0.5% agarose containing 10% FBS-DMEM, cell colonies were counted after 21-day incubation at 37 �C in 5% CO2 undermicroscopy. (B) Size >30 lM were counted as a colony. Each bar representsmean ± SD of triplicate experiments.

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We found that compound HTMC was the most potent amongtested, and caused a dose-dependent decrease in TA and hTERTmRNA expression without a marked alteration of hTR or GADD153as supported by both RT-PCR and Western blot experiments (Figs.2 and 3). It was interesting to note that HTMC, a natural chalconefrom a medicinal plant Caesalpinia pulcherrima,28 has significantcytotoxic activity against Jurkat (human lymphocytic) and U937(human monocytic) cells,21 however, the molecular mechanismsof its anti-proliferative action on malignant cell growth are not

clear. To the best of our knowledge, this was the first report to eval-uate the compounds tested here for their TA inhibitory assay.

hTERT is a structurally and functionally important component oftelomerase, which exerts its function of elongating telomere end re-peats in the nuclei. Telomerase inhibition is sequence-dependentand the primary mode of regulation of hTERT is proposed to be tran-scriptional.29 However, recent studies indicate that another possi-ble mechanism is the posttranscriptional regulation whichinvolved the translocation of hTERT from nuclei into cytoplasm.29

As shown in Figure 6E, immunofluorescence staining of HTMC trea-ted A549 cells demonstrated that the expression of hTERT proteindecreased. In addition, although hTERT is detected in both nucleiand cytoplasm, the majority of the signal is localized in the nuclei,as the production of hTERT takes place in cytoplasm, therefore aweaker hTERT staining can also be detected. These results indicatedthat HTMC regulation of hTERT was transcriptional in A549 cells. Inaddition, the �548 to +50 bp region proximal to the transcriptioninitiation site of the hTERT promoter is responsible for most of itstranscriptional activity. This region has several binding sites in-clude two typical E-boxes and several GC-boxes for the transcrip-tion factors c-myc and Sp1, respectively.2 The core promoterp548, which is necessary for hTERT expression also contains Sp1/Sp3 binding sites.2 Thus, investigation of mechanisms that modu-late hTERT expression in tumor and normal cells has become anarea of intense interest in the study of human cancer.30 In this re-port, the data from the luciferase assays showed that HTMC de-creased the hTERT promoter activity sequentially (Fig. 4). Theseresults further indicated that HTMC treatment in A549 cells in-duced the down regulation of TA with suppression of hTERT expres-sion at transcriptional levels. As the exact hTERT promoter-bindingsite of HTMC unknown, further studies were needed to elucidatethe effects of HTMC on the hTERT promoter activity.

Anchorage independence is an important feature that allows tu-mor cells to survive under certain inappropriate host conditions,such as during invasion and metastasis.31 In this report, A549 cellslost their ability to form colonies in the presence of HTMC for threeweeks treatment (Fig. 5). One possible explanation to the observedeffects was that HTMC-induced telomerase inhibition may cause achronic DNA damage response of an uncapped telomere or a fewchromosome ends thus reduced the colony formation efficiencyof the A549 cells.31

Figure 6. Involvement of intracellular calcium ion in 20-hydroxy-2,3,40 ,60-tetramethoxychalcone (HTMC)-induced inhibition of telomerase activity in A549 cells. (A) A549cells were treated with HTMC (0, 6.25, 12.5, 25.0 and 50.0 lM) for 24 h, and were treated with Fluo-3 AM for Ca2+ staining. (B) 1 h prior to incubation with HTMC, A549 cellswere pretreated with U0126 (10 lM), SB 203580 (50 lM), BAPTA-AM (20 lM), MG132 (20 lM), Leptomycin B (20 nM), LY294002 (50 lM). (C) Intracellular calcium chelatorBAPTA-AM (20 and 40 lM) was added to A549 cells 1 h prior to incubation with HTMC, after 24 h cells were harvested and subjected to TRAP assay. (D) Effect of BAPTA-AMon HTMC-induced suppression of hTERT mRNA level in A549 cells. BAPTA-AM (20 lM) was added to cells 1 h prior to incubation with HTMC and after 24 h total RNA wasisolated and RT-PCR was performed using indicated primers. The amplified products were run in 1.5% agarose gel and visualized by ethidium bromide staining. b-Actin wasused as an house-keeping control gene. (E) Effect of HTMC on the distribution of hTERT protein in A549 cells. Immunofluorescence staining for hTERT using specific anti-hTERT antibodies and TRITC goat anti-rabbit IgG (H+L) conjugate secondary antibodies in culture A549 cells after treatment with HTMC (25 lM) for 24 h.

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Calcium is an intracellular second messenger, which regulates avariety of biological processes.32 In most cases calcium does not actdirectly, but uses calcium-binding proteins as mediators of its sig-

nals then restored to basal level once signal is transmitted. Calciumbinds to these calcium-binding proteins and induces conforma-tional changes, thus exposing initially inaccessible protein binding

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sites and allowing their interaction with specific target proteins.32

Previously Rosenberger et al. (2007) suggest that calcium play afunctional role in the inhibition of TA in epidermal keratinocytes.33

These authors also reported that calcium-mediated inhibition oftelomerase activity by S100A8/S100A9 complex, in which calciumis bound to S100A9 and subsequently releases S100A8 from thecomplex, immediately S100A8 binds to telomerase, thus reducingtelomerase activity.33 In our study TRAP assay indicated that telo-merase activity is reduced in a dose-dependent and time-depen-dent manner (see Supplementary data Fig. 1). TA wassignificantly reduced at 25 lM HTMC, further analysis with thisconcentration at different treatment periods we found that telome-rase activity declined with longer treatment periods (see Supple-mentary data Fig. 1), this suggested once HTMC was taken in bycells, its telomerase inhibiting effect was a continuous process, be-sides its ability of reducing hTERT expression in RNA and proteinlevel (as shown in Fig. 3), it may also caused a direct inhibitory ef-fect by up-regulating calcium concentration. Therefore an in-creased calcium concentration can still be observed after 24 htreatment of HTMC. On the other hand, as shown in Figure 6A,treatment with HTMC increased calcium concentration in the nu-clei, this result suggested that calcium might play a role in themechanism of telomerase inhibition by HTMC. Previous reportindicate that a direct inhibition of telomerase activity by addingcalcium chloride in the cell-free extract by TRAP assay,33 and sup-ported our finding that focal calcium stimulation in nuclei byHTMC may inhibited TA via similar manner. BAPTA-AM is a cell-membrane permeable calcium chelator, which widely used as anintracellular calcium sponge. As shown in Figure 6A, calcium con-centration was increased within or around the nuclei area, whereasin A549 cells pretreated with BAPTA-AM, calcium stimulationswere seen in condensed, scattered dots outside the nuclei. Wehypothesize that calcium induced by HTMC was chelated by BAP-TA-AM which resulted restored the TA in cells treated with HTMC.These results further supported by the data from Figure 6B and C,in which, along with various other inhibitors, TA was only restoredin cells treated with BAPTA-AM, and supported our hypothesis thatcalcium participated in the mechanism of TA inhibition by HTMC.Taken together, these results demonstrated that HTMC inhibitedtelomerase activity through transcriptional regulation of hTERTand induction of Ca2+-dependent pathway.

4. Conclusions

In summary, this report identified a potent telomerase inhibitorHTMC. In addition, HTMC decreased the expression of hTERT, andsequentially reduced the hTERT promoter in A549 cells. HTMCtreatment also reduced the colony formation efficiency of A549cells. Additionally, the results of this study demonstrated that therelease of Ca2+ was the underlying mechanism of suppressed telo-merase activity and hTERT transcription. Therefore, the potential ofHTMC to inhibit telomerase activity and its associated molecularevents, and the chemical nature of this low-molecular-mass com-pound that translates to lower cost of synthesis compared to nucle-otide-based drugs suggested that it has potential to develop newtherapeutic drug for cancer therapy.

5. Experimental

5.1. Reagents and materials

Specialized chemicals used were obtained from Sigma ChemicalCompany (Saint Louis, MO). The preparation and structure identi-fication details of tested compounds are reported in our previouscommunications.21–26 Antibodies against hTERT, hTR, GADD153,

and substrate antibodies were purchased from Santa Cruz Biotech-nology (Santa Cruz, CA). Antibody against b-actin was obtainedfrom Sigma. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchasedfrom Promega (Madison, WI). Horseradish peroxidase-conjugatedgoat anti-rabbit secondary antibody (Cell Signaling, MA) and en-hanced chemiluminescence reagent (ECL) and Hyperfilm-ECL werepurchased from Amersham (Little Chalfont). All other reagentsused were of the analytical grade and obtained from Sigma.

5.2. Cell line and culture

Human lung adenocarcinoma cell line A549 was obtained fromthe American Type Culture Collection (Manassas, VA). Cells weremaintained at 37 �C in a 5% CO2-humidified atmosphere onDulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville,MD) and Basal medium Eagle (BME) (Sigma, Saint Louis, MI) mediumcontaining 10% fetal bovine serum (FBS) and 100 ng/mL each of pen-icillin and streptomycin (Life Technologies, Inc., Rockville, MD).

5.3. Telomere repeat amplification protocol (TRAP) assay

The TRAP assay was performed as we described previously.13

5.4. Cell proliferation assay

Cells were seeded at 1 � 107 cells/mL and then treated with theindicated concentrations of HTMC. After 24 h incubation, the cellnumber and viability were determined by MTS assay.13

5.5. Isolation of RNA and RT-PCR

The detailed steps of RNA isolation and RT-PCR were performedby the method as we described previously.13 The primer sequencesand PCR conditions for hTERT, hTR, GADD153 and b-actin were alsoperformed as we described previously.13 The products were visual-ized via electrophoresis on 1.5% agarose gel and stained with ethi-dium bromide. We confirmed the quality of cellular mRNAaccording to the intensity of b-actin.

5.6. Reporter gene assay

The hTERT promoter deletion mutants p548 (�548 to +50),p212 (�212 to +50), p196 (�196 to +50), and p177 (�177 to+50), cloned upstream of the firefly luciferase reporter in thepGL3-Basic vector (Promega Corp.) by following the protocol de-scribed previously.13 For luciferase assay, cells (7.5 � 104) wereseeded onto 24-well plates, cultured overnight, and transfectedwith the plasmids described above (1 lg/well) with DEAE-dextran(Amersham-Pharmacia plc, Little Chalfont, Bucks, UK). After 24 hincubation, the medium was carefully removed and fresh mediumcontaining various concentrations of tested compound was addedto the wells. The cells were treated continuously with HTMC for24 h. Cells were collected and transcriptional activity was assayedwith Luciferase Assay System (Promega). A plasmid expressing thebacterial b-galactosidase gene was co-transfected in each experi-ment to serve as internal control of transfection efficiency.

5.7. Western blot analysis

Anti-hTERT (ab32020, abcam) and anti-b-actin (AC-40, Sigma)were used for the detection of hTERT, and b-actin, respectively.The complete protocol for Western blot analysis has been de-scribed in our previous report.13 Blots were developed with an en-hanced luminol chemiluminescence (ECL) reagent (NEN, Boston).

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5.8. Soft agar colony formation assay

Anchorage-independent growth assay of A549 cells was per-formed by the method as we described previously.15

5.9. Fluorescence immunocytochemistry and confocalmicroscopy

A549 cells were seeded (1 � 104 cells per chamber) onto aneight-chamber slide (Nunc 177402, Naperville, IL). Cells were ren-dered quiescent, followed by treatment with HTMC for the indi-cated concentration. Cells were fixed in 4% paraformaldehyde for20 min and permeabilized with 0.3% Triton X-100 in PBS for20 min. Washed cells was incubated in 1% BSA in PBS for 1 h. Forco-immunostaining, cells were first incubated with an antibodyagainst hTERT (rabbit, 1:250; 1 h, room temperature; Rockland,Gilbertsville, PA), and tetramethylrhodamine isothiocyanate goatanti-rabbit IgG (H+L) conjugate was used as a secondary antibody(1:100, 1 h; ZyMax™ Grade, Invitrogen, Carlsboad, CA). Slides weremounted with mount medium and dried at room temperature. Nu-clei were counterstained with 0.2 lg of DAPI (40,6-diamidino-2-phenylindole) per mL. Computer-assisted image analysis of fluo-rescence was performed using a confocal microscopy scanning la-ser microscope (Leica TCS, wavelength excitation 488 nm, emission525 nm for FITC; 540/570 nm for TRITC).

5.10. Intracellular free calcium measurement

A549 cells were grown on poly-L-lysine coated glass coverslipsat 5 � 104 cells in 35 mm dish, and then treated with 25 lM HTMCfor 24 h. Other cells were pretreated for 1 h with MEK inhibitorU0126 (10 lM), or ERK inhibitor PD98059 (20 lM), or MAPKinhibitor SB203580 (50 lM), or intracellular calcium chelatorBAPTA-AM (20 lM), or 26S proteasome inhibitor MG132 (20 lM),or nuclear export inhibitor leptomycin B (20 nM), or PI3 K inhibitorLY294002 (50 lM) and then treated with 25 lM HTMC for 24 h.Cells were rinsed twice with HBSS (20 mM HEPES, 10 mM glucose,150 mM NaCl, 1.2 mM CaCl2, 5 mM KCl, 1 mM MgCl2, pH 7.4). Theywere then loaded with 3 lM Fura-3/AM dissolved in HBSS from aworking solution and 0.02% pluronic acid F-127, at 37 �C for60 min and then rinsed twice with HBSS. Next, the cells were incu-bated in HBSS for an additional 30 min to allow complete de-ester-ification of the dye. Calcium imaging was accomplished using anLSM 410 invert confocal laser scanning microscope (Carl Zeiss Jena,Germany). Excitation was done by the 488 nm line of an argon laser,emission was collected using a 505–550 band pass filter, and pin-hole was set at 1.87 airy units. Confocal imaging was performedwith a resolution of 512 � 512 pixel at 256 intensity. The frame ratewas 1 frame/min. Several cells were viewed together through 20�plan-Neofluar Zeiss objective (0.5 NA) using a factor-2 computerzoomed image. Detailed images were also collected using a 40�plan-Neofluar Zeiss objective (1.3 NA).

5.11. Statistics

The data are presented as mean ± standard deviation of tripli-cate experiments. Statistical comparisons were made by meansof one-way analysis of variance (ANOVA), followed by a Duncanmultiple-comparison test. The symbol (*) indicates p <0.05 whencompared with untreated controls.

Acknowledgement

This study was supported by National Science Council of Taiwan(NSC 98-2811-M-324-001 and NSC 99-2811-M-324-003).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmc.2010.08.021.

References and notes

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